1. Field of the Invention
The present invention relates generally to the fields of immunology and radiation oncology. More specifically, the present invention relates to radiolabeled fusion toxins for cancer therapy.
2. Description of the Related Art
Colorectal cancer is the second most common malignancy in the United States. It accounts for approximately 160,000 cases each year, which results in nearly 60,000 deaths (1). The treatment of metastatic disease remains particularly unsatisfactory. Many patients with colorectal cancer have disease that is at least microscopically disseminated when they are initially seen. Surgery can control their local disease if detected early, but because these cancers are relatively resistant to chemotherapy, another systemic treatment is needed to control metastatic disease. Although the introduction of intraarterial hepatic chemotherapy has led to an improvement in the disease-free survival of patients with colorectal cancer with metastatic disease limited to the liver (2,3), the survival of patients with metastatic colorectal cancer has improved little over the last 20 years.
Radiation therapy has been demonstrated to be an effective modality in the local and regional treatment of colorectal cancer when adequate doses are delivered. For instance, 4,500-5,000 cGy has been shown to control subclinical disease, and doses of 5,500-6,500 cGy can control gross disease in many instances (4,5). However, in the common setting of systemic metastatic disease, such doses cannot be safely delivered to the tumor because of the large volume of normal tissue that would be treated (4,5).
The use of external beam radiation therapy has produced curative treatment programs for several tumor types. However, this technique has practical limitations in regards to limited field of therapy, normal tissue toxicity, and radioresistance mechanisms. Considerable research efforts have been directed at ways to "target" radioactive isotopes to sites of malignant disease. Currently, the use of monoclonal antibodies (MoAbs) directed to "tumor-associated" antigens on cancer cells represents one approach which has had success in animal model systems (6-9) and is the subject of current phase I and II trials in man (10-15). Such a strategy provides the ability to localize radioactive isotopes to multiple sites of disease with hopefully adequate amounts of radiation to produce an antitumor effect and/or radioimmune imaging for diagnostic purposes. A second strategy is to use radioactively labeled peptides able to bind to receptor positive tumor cells, e.g. octreotide to somatostatin receptors in malignant carcinoid (16,17). Research efforts which provide better radioactive isotope delivery systems and/or targeting strategies will enhance the ability to apply targeted radiation therapy to human cancer.
Radiolabeled monoclonal antibodies (single-step radioimmunotherapy) have serious limitations in treating human cancer. Successful application of radiolabeled MoAbs in a single-step protocol for radioimmunodetection and radioimmunotherapy of tumors has been hindered in man by problems related to the low percentage uptake of injected radioactivity in tumors (0.001 to 0.1% ID/g), the slow penetration of relatively large (160 kDa) intact antibodies into tumors and heterogeneous distribution, their long persistence times in normal tissues leading to high background radioactivity and bone marrow suppression, and the development of human anti-mouse antibody (HAMA) responses. To overcome these problems, the use of antibody fragments and single chain antibodies (18-23), regional administration (24-26), the use of various radionuclides (6), the use of more stable (27) or enzymatically cleavable chelating agents (28), the use of cytokines to upregulate tumor-associated antigen expression (29,30), irradiation of the tumor to increase vascular permeability (15,31-33), the use of cytokines to protect against bone marrow suppression (34,35), and the use of autologous bone marrow transplantation (12,36) have been considered. Despite these efforts, the results of clinical radioimmunotherapy of solid tumors have been disappointing but antitumor efficacy has been demonstrated in clinical trials for therapy of the radiosensitive lymphoma types of tumors.
To maximize targeting molecule (e.g. MoAb) deposition into tumor sites while minimizing radioactive isotope exposure to the bone marrow, investigators have designed strategies to separate these two components. One strategy has been to develop bifunctional MoAbs with one combining site for tumor and a second binding site for the radioactive ligand (37-47). The bifunctional antibody is administered and allowed to circulate for several days (optimal tumor deposition) and then the radioligand is administered with a rapid tissue distribution and short plasma half-life. This strategy allows tumor localization of the isotope to occur rapidly (matter of a few hours) with limited radiation doses to the bone marrow. The major limitation of this strategy has been the reduced affinity of the individual antigen combining sites (single rather than dual binding sites similar to Fab fragments), variable kinetics/distribution of the separate components making optimal schedules of therapy difficult to standardize, and normal tissue extravascular distribution of the targeting molecule.
A second approach is to take advantage of the high affinity avidin-biotin system by conjugating one of the pair to a MoAb for targeting and the other member of the pair in a radioligand preparation. In this way, the MoAb retains its affinity for tumor antigen while the short-lived radioligand has high binding affinity to the MoAb conjugate. Systems using either streptavidin radioligand (37,48-53) or biotin radioligand (37,48,54,55) have been described. These studies have generally used radioactive metal chelates (.sup.111 In, .sup.90 Y and .sup.186 Re) but Del Rosario and Wahl (56) developed a multivalent biotinylated radioiodinated polylysine peptide as well. A major drawback of this system is that the high affinity binding of radioligand to MoAb conjugate occurs with any residual MoAb in the plasma or extravascular space. The use of an additional step (39,49,57) to clear circulating antibody conjugates improves the distribution of the radioligand but results in a complex and variable schedule of MoAb conjugate infusion, plasma conjugate clearing reagent administration, and radioligand infusion.
The physical and chemical properties of a radionuclide are important in its selection for radiotherapy, e.g., the type of particulate emission must be considered (58). The potent lethality of Auger and low-energy conversion electrons has been demonstrated (59-62). This effect can best be realized with intranuclear localization of the radionuclide, which does not generally occur with radiolabeled MoAbs, but may occur with certain membrane receptor-radioligand interactions. Of course, alpha particles have a high linear energy transfer (LET) effective in cell killing and a range of several cell diameters, 40-80 .mu.m. They may have a role for therapy of micrometastases, leukemia, and intracavitary administration (180). Beta particles are less densely ionizing and have a range longer than alpha particle emitters so that the tumor distribution requirements are less restrictive. On the other hand, for micro-metastases, the absorbed fraction for higher energy beta particles (range&gt;tumor size) is decreased, leading to a less favorable tumor absorbed dose. The gamma-ray energies and abundances are also important physical properties, because the presence of gamma rays offers the possibility of external imaging but also adds to the whole-body radiation dose.
These physical and chemical factors must then be viewed in light of available biological information (58,63). There is substantial variation in radioligand uptake, macro- and micro-distribution, kinetics and metabolism/catabolism depending on the particular radioligand, radioligand dose, the variability of antigen/receptor expression in the tumor, its size and stage, etc. (64-77). This may be due to cell type heterogeneity, heterogeneity of antigen/receptor expression, heterogenous vascularity and capillary permeability, elevated interstitial pressure, the binding site barrier, and spatial inaccessibility (15,58,64-78). The expected nonuniform distribution of radioligand discussed above reduces the attractiveness of short-ranged alpha-emitting radionuclides for solid tumor radiotherapy. A role for alpha emitters may be feasible in specific cases such as for micrometastases or intracavitary administration for some types of cancers, such as peritoneal injection for colorectal or ovarian carcinoma (58,79,80). The longer range of beta particles can still permit uniform tumor irradiation despite a marked heterogeneity of distribution of radioactivity within the tumor. It appears desirable to deliver ionizing radiation with a range of one to several millimeters in solid tumors, as from intermediate to high-energy beta particles.
Beta emitters offer a wide choice of candidates with a selection of particle ranges and chemical properties. The use of radionuclides with some gamma emission would allow diagnostic low-dose experiments to determine biodistribution prior to administering a therapeutic dose of the exact same preparation.
Most therapeutic trials to date using radioimmunotherapy have utilized .sup.131 I, largely due to its ready availability at moderate cost, the ease of radioiodination techniques for proteins, and its long history of use in treating thyroid malignancy, rather than any careful analysis of its suitability for radioimmunotherapy. .sup.131 I has a physical half-life of 8.04 days, maximum beta energy of 0.6 MeV, average beta energy of 0.2 MeV, and is considered a medium-range beta emitter (mean range about 500 .mu.m) with a maximum range of 1.5 mm in soft tissue. However, the yield of penetrating gamma radiation with .sup.131 I (average energy of 0.36 MeV) constitutes two-thirds of the total absorbed dose equivalent of this source in humans, resulting in higher total body doses away from the tumor volume thereby contributing to bone marrow toxicity. There is also a problem with dehalogenation producing a further loss in specific targeting, retention in tumor, and an increase in toxicity.
.sup.90 Yttrium is being studied as a radioimmunotherapy isotope (6,15,78, 81-88) because of its characteristics which include a 64 h half-life and an intermediate beta energy (2.3 MeV maximum). Since .sup.90 Y is unsuitable for quantitative imaging, .sup.111 In biodistribution data can be used to predict dose for .sup.90 Y administrations (81,82). However, even though there are similarities in tumor uptake, blood clearance and normal tissue uptake, there may be substantial differences in retention and clearance from kidney, bone, and the reticuloendothelial system.
.sup.186 Rhenium has some attractive features for radioimmunotherapy. The energy contribution from gamma rays of .sup.186 Re is 137 keV with only 8.65% abundance, which should result in a lower dose to the whole-body than with .sup.131 I. The gamma radiation from .sup.186 Re is high enough to be efficiently used for external imaging. Finally, the x-rays from .sup.186 Re are low energy radiations (59-73 keV, 9.2% abundance) and there is only a small contribution from this source to the whole body dose. Even though imaging photons in .sup.186 Re can be used particularly at therapeutic dose levels (89,90) the "matched pair" approach using .sup.99m Tc and .sup.186 Re (the former for imaging and the latter for therapy) is a very attractive option (58,90). These can both be attached to antibodies via similar chemistry (58,91) and generally produce similar biodistributions. Rhenium-186 requires a high flux reactor to achieve adequate specific activity and it is available commercially.
Copper-64 is a positron emitting isotope (.beta.+max=656 keV; 19% abundance) that has been used for diagnostic imaging. However, it also has a 573 keV maximum .beta..sup.- emission (38% abundance) and has been shown to be effective as a therapeutic radionuclide. This isotope has a 12.8 hour half-life, making it ideal for labeling peptides which have a short tumor localization time. Copper-64 is available from the University of Missouri Research Reactor (MURR) on a bimonthly basis in high specific activity (4,000-10,000 Ci/mmol). Copper-67 is a pure beta emitter (P-max=570 keV) with a 62 h half-life and has been used in several therapy studies. It also has a gamma emission of 185 keV (40% abundance) for imaging. It can be purchased from Brookhaven National Laboratory or Los Alamos National Laboratory; however, it is of much lower specific activity than .sup.64 Cu. Protracted lysosomal retention following cellular internalization has been reported for .sup.67 Cu.
Alpha particles and other heavy particles interact with matter producing dense trails of ionization. This effect, known as high linear energy transfer (LET), produces a greater relative biological effectiveness (RBE) than low LET radiation, principally photons and electrons. When faced with a limited number of receptors on cells, this improves the cytotoxic efficacy with the high LET emitters.
There are two additional important advantages of high LET radiation that are important for their use in radiotherapy. The first is the independence of cytotoxicity from the rate at which dose is delivered. Dose rates typically quoted for radioimmunotherapy are on the order of 20 cGy/hour or lower. These dose rates have been shown to be suboptimal in their cytotoxic effect on adenocarcinomas for low LET radiation (94). Implant therapy and brachytherapy strive to maintain dose rates of 40 cGy/hour or greater to minimize the dose rate effects (95). By comparison, dose delivered in a clinical radiation therapy external beam unit is in the range of 12,000 cGy/hour. High LET emitters have the same cytotoxicity independent of the dose rate (96,97). This is critical for radiotherapy where the dose rate begins low and is continuously decreasing. In some systems, like hepatoma (194), low dose rate does not seem to be the limitation that it is in adenocarcinomas.
The second advantage of high LET emitters is their cytotoxicity in the absence of oxygen (96). Low LET radiation requires the presence of oxygen to form free radicals that inflict the damage to the cell components that result in cell killing. Because high LET radiation is so densely ionizing, the free radicals can be formed directly and do not require the presence of oxygen. This is an advantage of high LET radiation in treatment of tumors that have areas of hypoxia. The ability to kill cells in these regions with the same dose of radiation is an advantage in therapy.
One of the disadvantages of high LET emitters for radiotherapy has been the limited selection of appropriate radionuclides. Given constraints on the half-life, photon emission and stability of daughter products, there are few candidate radionuclides for therapy. One radionuclide, Astatine-211, has the disadvantage of requiring a cyclotron that can accelerate He-4 ions in order to produce it. This, coupled with its 7.2 hour half-life, creates serious problems in supply.
With this in mind, one investigator developed a new generator system that is capable of producing Lead-212 (98). Lead-212 has a 10.6 hour half-life and decays by beta emission to .sup.212 Bi. Bismuth-212 has a 1 hour half-life and decays by beta and alpha emission to stable .sup.208 Pb. This system combines some of the most attractive aspects of radionuclides for therapy. Lead-212 is produced from Radium-224 which has a 3.6 day half-life, on the order of Molybdenum-99. The .sup.212 Pb can be generated on site in a no-carrier-added form ideal for conjugating to small amounts of reagent, thus insuring high specific activity. The half-life is ideal for therapy, long enough to insure that localization can occur, but short enough to not present serious problems in redistribution in vivo.
The advantages of high LET radiation have been exploited by using alpha-emitting radionuclides for therapy (99-104). Bloomer et al. (99) used a colloid to keep .sup.211 At in the peritoneal cavity with the hope that the superior cytotoxicity of this radiopharmaceutical would replace .sup.32 P colloid. Link and Carpenter tested a small molecule that was chemically attached to .sup.211 At (100). The combination of monoclonal antibodies and high LET emitters, directing the radiation to the tumor cell surface, has been studied (101-104). In this case, the short range of the alpha particle in vivo necessitates keeping the emission near the cell surface or inside the cell (105). Subsequently, a .sup.211 At-labeled antibody was used to treat a meningioma successfully (101).
Gansow et al. have experimented extensively with chelate systems that could be used to radiolabel antibodies with .sup.212 Bi and its parent, .sup.212 Pb (106-109). They have found that a derivative of DTPA can bind .sup.212 Bi effectively but this chelate does not bind .sup.212 Pb. DOTA, a macrocycle that contains amine and carboxylate groups, can be used to bind the .sup.212 Pb.
Little is known about the destructive effects of nuclear transformation of beta emitters in aqueous solutions. In a recent in vitro study of the chemical fate of .sup.212 Bi(DOTA).sup.1- formed by beta decay of .sup.212 Pb(DOTA).sup.2-, the fraction of .sup.212 Bi radioactivity not complexed to DOTA was found to be 36.+-.2% (107). Analysis of various processes responsible for excitation of the .sup.212 Bi daughter, breakup of the .sup.212 Bi-DOTA complex was ascribed to the internal conversion of gamma rays emitted by the excited .sup.212 Bi nuclide. In the case of conventional, directly labeled antibody or fusion toxin in which the majority of the protein is in the circulation, the released .sup.212 Bi is bioavailable to localize to normal organs. In a control biodistribution study of .sup.212 Bi, high levels were found in the kidney (110). In the intraperitoneal model, tumor localization and internalization takes place within the first few minutes following injection, before significant decay to .sup.212 Bi can take place. Thus, release of the .sup.212 Bi in the peritoneum should be limited considering the relatively short half life of .sup.212 Bi and uptake in normal organs may be minimal.
Targeted therapy with immunotoxins has serious limitations in treating solid cancers. Immunotoxins (IT) are a class of pharmacological reagents produced by conjugating MoAbs to potent catalytic toxins (111,114-118) which should selectively bind to and kill cancer cells while not harming normal cells. The first generation of such IT were conjugates of MoAbs to toxins such as ricin, diphtheria toxin (DT), or Pseudomonas exotoxin (PE). The plant toxin ricin, one of the most toxic substances known, is a lectin with specificity for galactose terminating glycoproteins. Ricin consists of a 30 kDa A chain and a 30 kDa B chain joined by a disulfide bond. The A chain subunit is an enzyme which inactivates 28S components of the 60S subunit of ribosomes. The B chain subunit binds whole ricin to native receptors on the cell surface. The native receptors are galactose-containing glycoproteins that are present on all eukaryotic cells. The conjugation of A chain alone to MoAbs circumvents the risk of nonspecific binding. Intact ricin conjugates have limited utility in vivo. This is attributable to the galactose binding site of ricin having a high affinity for the galactose receptor on the cell surface of normal cells. IT consisting of ricin A chain conjugated to MoAbs with reactivity against human colon cancer have been synthesized and shown to inhibit protein synthesis and to be cytotoxic to human colon cancer cells in vitro (119-126), and to inhibit the growth of human tumor xenografts (127). Investigators have constructed IT that contain B chain with its native binding site chemically blocked (125,128-130). The obstruction of the B chain binding site is meant to prevent nonspecific toxicity to normal cells. Cattel et al. (125) reported that blocked intact ricin B chain IT displayed more selective cytotoxicity to target human colorectal adenocarcinoma cell lines than non-blocked IT and was much more potent than the ricin A chain IT.
Although there are reports describing the in vitro killing of tumor cells by IT, there are few papers reporting the successful use of IT against carcinomas in vivo. IT with reactivity against human colon and pancreatic tumors have been synthesized (119-127,131-133). Other IT have been used in therapeutic studies in animal tumor xenograft models (reviewed in 134,135) and recently in clinical studies in patients with leukemia, breast cancer, and melanoma (136-140). Initial clinical trials have not thus far shown significant activity of IT in patients with solid tumors (112,141,142). A pattern of mixed regression, which is defined as a 50% or greater reduction in size in one or more metastases combined with an increase in the size of one or more concurrent lesions or the appearance of a new lesion after the initiation of therapy, occurred in a minority of patients with metastatic malignant melanoma and colorectal cancer treated with IT specific for each of these cancers (141). Clinical responses in patients with hematologic malignancies have been more encouraging. In patients with B-cell lymphoma, IT containing MoAbs reactive with B-cell antigens coupled to either ricin A chain or blocked ricin produced partial regressions, which are defined as a 50% or more reduction of the overall tumor burden, in about 40% of patients (141). Problems in therapeutic applications, particularly toxicity, make it clear that simply conjugating an antibody to a toxin will not cure cancer. Some of the antibodies used for the preparation of IT to treat solid tumors had cross-reactivity with neural tissue or bone marrow which resulted in toxicity to these tissues (142).
In the clinical trials using ricin-based IT, the nonspecific toxicity of the toxin moiety remains the major problem (112). Capillary leak syndrome and liver dysfunction are the limiting toxicities for deglycosylated ricin A chain and blocked ricin, respectively (112). With PE, the dose-limiting toxicity is damage to the liver. The inability to deliver repeated doses of IT to patients due to antibody formation is a major concern. Although the formation of HAMA can be avoided by the use of humanized antibodies or humanized forms of single-chain Fv-IT, the problem of formation of antibodies against the toxin moiety remains unsolved (112). Antibodies to DT already exist in most individuals who received immunizations with diphtheria, pertussis, and tetanus. These factors may limit the therapeutic efficacy of IT.
Recombinant fusion toxins may be useful for therapy of solid cancers. Progress in the knowledge of the structure and function of several toxins, advanced cloning and preparation technologies, and the development of recombinant antibodies has made it possible to construct new kinds of IT. Protein toxins such as PE, DT, and ricin may be useful in cancer therapy because they are among the most potent cell-killing agents. The genes encoding these three toxins have been cloned (112,145) and expressed in Escherichia coli (E. coli). Both DT and PE inhibit protein synthesis by catalytically inactivating elongation factor 2 in the cytosol which is necessary for protein synthesis (111,145,146). To get to the cytosol, toxins must first bind to the target cell, be internalized by endocytosis, and translocate to the cytosol. Only a few molecules need reach the cytosol to kill a cell. A major advance in the IT field has been the production of recombinant fusion toxins (111,145,146); these were created by fusing DNA elements encoding binding regions of antibodies, growth factors, or cytokines to a mutant form of a toxin gene. Recombinant fusion toxins bind to cells, undergo endocytosis, and kill cells that are recognized by the antigen- or receptor-binding domain. Although these molecules are not IT in a strict sense, because they do not contain an antibody moiety, they are closely related to IT in their mode of targeting and killing of cells.
A number of recombinant fusion toxins have been developed recently for use in receptor-targeted cancer therapy (111,112,145,146). Many tumors express higher levels of growth factor receptors or antigens than normal cells, and often they express receptors for more than one ligand. These genetically engineered chimeric fusion toxins have been generated by deleting the portion of the toxin gene encoding the cell binding domain and replacing it with DNA encoding an alternative cell binding peptide such as a growth factor, lymphokine, or single chain antibody.
In 1988, it was shown that recombinant forms of antibodies consisting of variable domains of the heavy and light chains linked together via a peptide linker were able to bind antigen (111). Such proteins were termed single chain antibodies, or Fv domains. Anti-Tac(Fv) reactive with the interleukin-2 receptor (IL-2R) was fused to a mutant form of PE called PE40 in which the binding domain of the native toxin has been deleted (111,146). This recombinant IT killed leukemia cells in vitro.
DT is a single-chain polypeptide of 535 residues produced by Corynebacterium diphtheriae. DT and recombinant forms of DT have been coupled to antibodies, growth factors, and cytokines and have been shown to have cytotoxic activity in vitro. The receptor-binding domain of native DT has been replaced with IL-2, the Fv region of anti-Tac antibody, IL-4, IL-6, or EGF. The resulting recombinant fusion toxins are selectively toxic to cells that express the specific receptor.
Limited transvascular diffusion of IT into solid tumors represents a problem. Penetration is mainly dependent on size; the smaller the IT, the better its tumor penetration (111). Affinity also influences tumor penetration (111). Fv fragments of antibodies (22 kDa) penetrate tumors better than Fab fragments (45 kDa) which in turn penetrate better than intact antibodies (188). Due to their larger size, the ability of IT to penetrate into solid tumors by diffusion and convection is impaired, although they have a half-life in the circulation of 4-8 hours or more. Therefore, first-generation IT (200 kDa) should take much longer to penetrate a 1 cm diameter solid tumor than a recombinant fusion toxin (60 kDa). Tumor penetration is much more effective for small recombinant fusion toxins, but they are also cleared very rapidly from the circulation (T.sub.1/2 =20-40 minutes).
The prior art is deficient in the lack of effective means of inhibiting the growth of various cancers. The present invention fulfills this longstanding need and desire in the art.